Chromatographic methods are commonly used to separate a mixture into various components, so that these components can be identified and quantified. In one class of chromatographic methods, mixtures are transmitted along a separation column through which the various components migrate at different flow velocities. Such techniques are applied to gaseous samples as well as to liquid samples. These techniques are now widely utilized in chemical, biological and medical applications.
A variety of mechanisms are utilized to produce the desired separation between the components of the mixture. In one class of systems, the wall of the tube through which the mixture flows is coated with a material that exhibits different affinities for different components of the mixture. The speed of migration along the channel is greater for components exhibiting a weaker affinity for the wall, thereby producing a separation of mixture components according to their affinity for the coated wall.
In another class of systems, the column is packed with a material, such as a gel, that provides the differential interaction with the components of the mixture. The packing functions as a sieve that physically retards the flow of larger molecules more than it retards the flow of smaller molecules. In addition, the surface of the packing can contain chemical groups that control the affinity between the packing and various components of the mixture. Therefore, the wide range of choices of pore size and surface groups makes packings, such as gels, a very flexible medium for separating the components of the mixture. The sample fluid can be forced through this packing by a number of techniques, such as a pressure difference between the inlet and outlet ends of the capillary (also referred to herein as a "column") and/or the use of capillary electrophoresis to drive sample ions through the packing.
These separation columns typically pass the sample solution past a detector that measures some physical property of the components, such as the absorbance spectrum, the fluorescence spectrum, the refractive index or the electrical conductivity of the sample solution. In each of the first three of these particular cases, an optical beam is passed through the sample. For the case of absorbance measurements, a detector is positioned along a straight line through the optical source and the separation column to receive the optical beam after it passes through the sample. Fluorescence detection exhibits the advantages of superior selectivity and sensitivity in detecting many compounds.
For the case of fluorescence measurements, the location of the detector is determined by considerations of the signal-to-noise ratio of the output of the detector. As taught in U.S. Pat. No. 4,548,498 entitled Laser Induced Fluorescence Detection In Modem Liquid Chromatography With Conventional And Micro Columns issued to Folestad, et al on Oct. 22, 1985, the incident beam of light is typically perpendicular to the axis of the capillary and therefore scatters off of the wall of the capillary into a plane oriented perpendicular to the axis of the capillary and passing through the point of the capillary illuminated by the incident beam of light. To avoid such scattered light in the case of fluorescent light detection, the detector is positioned outside of this plane and oriented to receive light from the sample along a direction at 30.degree. with respect to the plane of scattered light. U.S. Pat. No. 4,675,300 entitled Laser Excitation Fluorescence Detection Electrokinetic Separation issued to Zare, et al on Jun. 23, 1987 teaches that the use of a coherent light has the advantage of reducing the Raman and Rayleigh scattering components of the scattered light.
Conventional flowcells for fluorescence detectors for capillary liquid chromatography often use a lens to focus excitation light onto the liquid column and often use a lens to focus fluorescent light onto the detector. In the capillary flowcell presented in the article On-Column Capillary Flow Cell Utilizing Optical Waveguides For Chromatographic Applications by Alfredo E. Bruno, et al, Anal. Chem. 1989, 61, p. 876-883 optical fibers are utilized to carry incoherent light to the flowcell and are also utilized to transmit fluorescent light from the flowcell to a detector.
This article further teaches that optical fibers are as efficient as laser sources in directing the exposing light through the bore of the capillary to the capillary flowcell. For a capillary of specified inner and outer diameters, a ray path calculation is presented that enables a determination of the maximum diameter of the source optical fiber and the minimum diameter of the collecting optical fiber needed to pass substantially all of the source light through the bore of the capillary to the collecting optical fiber. An analysis of the distribution of scattered light and the fraction of incident light that actually passes through the bore of the capillary is also presented in this article. Because there is no focussing of the light from the capillary onto the collecting optical fiber, the collection efficiency of the collecting optical fiber is limited by its acceptance angle and the spacing between the capillary input end of this collecting optical fiber. Typically, each of these fluorescent systems collects less than one-eighth of the fluorescent light emitted from that system.
U.S. Pat. No. 4,199,686 entitled Dark Field Illuminator And Collector Apparatus And Method issued to Brunstig, et al on Apr. 22, 1980 and U.S. Pat. No. 4,273,443 entitled Method And Apparatus For Measurement Of Reradiation In Particle Flow Cell Systems issued to Walter P. Hogg on Jun. 16, 1981 present optical sections of a device for counting particles, such as biological cells. Although this optical section is efficient at collecting fluorescent light emitted by the particles exposed by the incident light, it just as efficiently collects the light scattered by these particles as well as the light that passes through the particle stream without being scattered or absorbed by the particles. Therefore, some mechanism, such as a dichroic filter or a Fresnel prism, is included to deflect the nonfluorescent portion of the light focussed by the optical section onto the detector. The need for this mechanism increases the complexity and cost of this system. Furthermore, to the extent that this mechanism is not 100% efficient in discriminating against such nonfluorescent portion of the collected light, this structure will exhibit a reduced signal-to-noise ratio.
U.S. Pat. No. 4,088,407 entitled High Pressure Fluorescence Flow-Through Cuvette issued to Dietmar M. Schoeffel, et al on May 9, 1978, presents a cuvette for liquid chromatographic analysis. A collector, formed as a reflective coating on a surface that is parabolic, circular or elliptical surface or revolution, redirects part of the fluorescent light onto a detector. Because of the large size of the chamber into which the sample liquid is directed for fluorescent excitation, this structure is unsuitable for use with capillary-based systems such as capillary liquid chromatography and capillary electrophoresis, because it would introduce a completely unacceptable amount of band broadening into the chromatic separation process. Because the specification lacks any dimensional details regarding the collector and the width of the beam of exposing light, it is impossible to determine the efficiency of this structure for collecting the fluorescent light from the exposing region of the sample liquid.
Because the use of small diameter bore capillaries improves the separation between the components of a sample, capillary liquid chromatography uses columns with very small internal diameters (typically 5 to 300 microns). Because of this, only a very small quantity of sample (on the order of 1-2 nanoliters) is exposed at any given time during a measurement. Therefore, the signal to noise ratio (referred to as the "S/N ratio") for such systems is very dependent on: the fraction of fluorescent light from the sample that is actually collected by a system detector; and the amount of stray light that reaches the detector. A major limitation in the use of fluorescence detection has been the achievable signal to noise ratio.
The S/N ratio has been improved by use of stronger light sources and/or coherent light sources in which the energy is concentrated at a wavelength that is particularly efficient at producing fluorescence. This improvement therefore arises from an increased amplitude of the signal. However, part of the noise component, such as scattered light, is proportional to the intensity of the exposing light. Therefore, when this portion of the noise is a significant fraction of the total noise, it is important to minimize such component. It is therefore very important to utilize a structure that prevents substantially all stray light from reaching the detector, so that the noise component is minimized. This means that care must be taken to minimize the amount of scattered light that is produced and to ensure that substantially none of this scattered light reaches the detector. It is also important to ensure that a very high fraction of the fluorescent light emitted by the sample reaches the detector, so that the signal component of the detector output is substantially maximized.